Skip to main content
Log in

Diversity of culturable denitrifying bacteria

Limits of rDNA RFLP analysis and probes for the functional gene, nitrite reductase

  • Original Paper
  • Published:
Archives of Microbiology Aims and scope Submit manuscript

Abstract

Sequence divergence in the ribosomal genes of known strains and isolates of aquatic denitrifying bacteria was investigated using restriction fragment length polymorphism (RFLP) analysis. The same cultures were characterized for their homology with antibody and gene probes for nitrite reductase (NiR), a key enzyme in the denitrification pathway, and for amplification with a set of polymerase chain reaction primers designed to amplify a portion of the NiR gene. The NiR probes were developed from Pseudomonas stutzeri (ATCC 14405) and several P. stutzeri strains were included in the analyses. The RFLP analysis clustered most of the P. stutzeri strains together, but detected considerable diversity within this group. Isolates from three aquatic environments exhibited within —and among — habitat diversity by RFLP. Hybridization with the NiR probes and amplification with the NiR primers were not correlated with the clustering of strains by rDNA RFLP analysis. The relationships among strains deduced from ribosomal DNA RFLP reflect heterogeneity within the P. stutzeri group and among other pseudomonads, and the patterns differ from those inferred from specificity of the NiR probes.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Similar content being viewed by others

Abbreviations

NiR :

Nitrite reductase

PCR :

polymerase chain reaction

RFLP :

restriction fragment length polymorphism

References

  • Ausubel FM, Brent R, Kingston RE, Moore DD, Smith JA, Sideman JG, Struhl K (eds) (1987) Current protocols in molecular biology. Wiley, New York

    Google Scholar 

  • Baumann L, Baumann P, Mandel M, Allen RD (1972) Taxonomy of aerobic marine eubacteria. J Bacteriol 110:402–429

    Google Scholar 

  • Bendschneider K, Robinson RJ (1952) A new spectrophotometric method for the determination of nitrite in seawater. J Marine Res 11:87–96

    Google Scholar 

  • Biebl H, Pfennig N (1978) Growth yields of green sulfur bacteria in mixed cultures with sulfur and sulfate reducing bacteria. Arch Microbiol 117:9–16

    Google Scholar 

  • Brosius J, Ullrich A, Raker MA, Gray A, Dull TJ, Gutell RR, Noller HF (1981) Construction and fine mapping of recombinant plasmids containing the rrnB ribosomal RNA operon of E. coli. Plasmid 6:112–118

    Google Scholar 

  • Bryan BA (1981) Physiology and biochemistry of denitrification. In: Delwhich CC (ed) Denitrification, nitrification and atmospheric nitrous oxide. Wiley, New York, pp 67–84

    Google Scholar 

  • Carlucci AF, Pramer D (1957) Factors influencing the plate method for determining abundance of bacteria in sea water. Proc Soc Exp Biol Med 96:392–394

    Google Scholar 

  • Coyne MS, Arunakumari A, Averill BA, Tiedje JM (1989) Immunological identification and distribution of dissimilatory heme cd 1 and nonheme copper nitrite reductases in denitrifying bacteria. Appl Environ Microbiol 55:2924–2931

    Google Scholar 

  • Denamur E, Picard B, Decoux G, Denis J-B, Elion J (1993) The absence of correlation between allozyme and rrn RFLP analysis indicates a high gene flow rate within human clinical Pseudomonas aeruginosa isolates. FEMS Microbiol Lett 110:271–280

    Google Scholar 

  • Döhler K, Huss VAR, Zumft WG (1987) Transfer of Pseudomonas perfectomarina Baumann, Bowditch, Baumann, and Beaman 1983 to Pseudomonas stutzeri (Lehmann and Neumann 1896) Sijderius 1946. Int J Syst Bacteriol 37:1–3

    Google Scholar 

  • Gamble N, Betlach M, Tiedje JM (1977) Numerically dominant denitrifying bacteria from world soils. Appl Environ Microbiol 33:926–939

    Google Scholar 

  • Jungst A, Wakabayashi S, Matsubara H, Zumft WG (1991) The nirSTBM region coding for cytochrome cd 1 dependent nitrite respiration of Pseudomonas stutzeri consists of a cluster of mono-, di- and tetraheme proteins. FEBS Letters 279, 205–209

    Google Scholar 

  • Körner H, Frunzke K, Dohler K, Zumft WG (1987) Immunochemical patterns of distribution of nitrous oxide reductase and nitrite reductase (cytochrome cd 1) among denitrifying pseudomonads. Arch Microbiol 148:21–24

    Google Scholar 

  • Liesack W, Weyland H, Stackebrandt E (1991) Potential risks of gene amplification by PCR as determined by 16S rDNA analysis of a mixed-culture of strict barophilic bacteria. Microbial Ecol 21:191–198

    Google Scholar 

  • Navarro E, Simonet P, Normand P, Bardin R (1992) Characterization of natural populations of Nitrobacter spp. using PCR/RFLP analysis of the ribosomal intergenic spacer. Arch Microbiol 157:107–115

    Google Scholar 

  • Nei M, Li W-H (1979) Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc Natl Acad Sci USA 76:5269–5273

    Google Scholar 

  • Palleroni NJ, Doudoroff M, Stanier RY, Solanes RE, Mandel M (1970) Taxonomy of the aerobic Pseudomonads: the properties of the Pseudomonas stutzeri group. J Gen Microbiol 60:215–231

    Google Scholar 

  • Rosello R, Garcia-Valdes E, Lalucat J, Ursing J (1991) Genotypic and phenotypic diversity of Pseudomonas stutzeri. System Appl Microbiol 14:150–157

    Google Scholar 

  • Rosello R, Garcia-Valdes E, Lalucat J, Ursing J (1993) Taxonomic relationship between Pseudomonas perfectomarina ZoBell and Pseudomonas stutzeri. Int J Syst Bacteriol 43:852–854

    Google Scholar 

  • Smith GB, Tiedje JM (1992) Isolation and characterization of a nitrite reductase gene and its use as a probe for denitrifying bacteria. Appl Environ Microbiol 58:376–384

    Google Scholar 

  • Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517

    Google Scholar 

  • Sylvestrini MC, Galeotti CL, Gervais M, Schinina E, Barra D, Bossa F, Brunori M (1989) Nitrite reductase from Pseudonomas aeruginosa: sequence of the gene and the protein. FEBS Lett 254:33–38

    Google Scholar 

  • Ward BB, Cockcroft AR (1993) Immunofluorescence detection of denitrifying bacteria in seawater and intertidal sediment environments. Microbial Ecol 25:233–246

    Google Scholar 

  • Ward BB, Cockcroft AR, Kilpatrick KA (1993) Antibody and DNA probes for detection of nitrite reductase in seawater. J Gen Microbiol 139:2285–2293

    Google Scholar 

  • Woese CJ (1987) Bacterial evolution. Microbiol Rev 51:221–271

    Google Scholar 

  • Zumft WG (1992) The denitrifying prokaryotes. In: Albert Ballows et al. (eds) The prokaryotes, 2nd edn. Springer, New York, pp 554–582

    Google Scholar 

  • Zumft WG, Gotzmann DJ, Kroneck PMH (1987) Type 1, blue copper proteins constitute a respiratory nitrite-reducing system in Pseudonomas aureofaciens. Eur J Biochem 168:301–307

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ward, B.B. Diversity of culturable denitrifying bacteria. Arch Microbiol 163, 167–175 (1995). https://doi.org/10.1007/BF00305349

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00305349

Key words

Navigation